Artificial light at night bans Chaoborus from vital epilimnetic waters

Artificial light at night (ALAN) is known to affect organisms in terrestrial ecosystems and adjacent litoral habitats. In the present study, we tested the effect of ALAN on the spatial distribution of organisms in open waters, using the insect larvae of Chaoborus flavicans as an example. During the day C. flavicans typically hide from visually hunting fish in deep, dark, anoxic waters. On safer nights, they forage in rich subsurface waters. Nighttime field tests revealed that light from an HPS street lamp mounted on a boat anchored in open water attracted planktivorous fish, but deterred planktonic Chaoborus from rich but risky surface waters. Chaoborus did not descend to the safest, anoxic hypolimnion, but remained in hypoxic mid-depth metalimnion, which does not appear to be a perfect refuge. Neither light gradient nor food distribution fully explained their mid-depth residence under ALAN conditions. A further laboratory test revealed a limited tolerance of C. flavicans to anoxia. Half of the test larvae died after 38 h at 9 °C in anoxic conditions. The trade-off between predation risk and oxygen demand may explain why Chaoborus did not hide in deep anoxic waters, but remained in the riskier metalimnion with residual oxygen under ALAN conditions.


Natural distribution of the Chaoborus and fish
The diurnal pattern of the spatial distribution of the Chaoborus larvae and fish recorded by sonar in the lake Roś in 2015 (Fig. 1) is a typical distribution of those animals in eutrophic lakes with anoxic hypolimnion in summer .At midday most Chaoborus resided deep (20-25 m) in the water column a few meters above the bottom in cold, dark anoxic waters.Fish were not visible on the echogram in that strata, at that time.Fish in shoals were visible in the epilimnetic waters in the further daytime hours.At dusk, pelagic shoals of fish dispersed and density of fish in the epilimnetic waters increased most likely due to horizontal migration from the littoral, while Chaoborus larvae rose toward the surface.At 8:00 p.m. and 9:00 p.m. Chaoborus larvae and fish were still separated vertically, but later on, as light intensity further decreased, they started to share the epilimnetic zone, the co-occurrence in the surface water lasted from 10:00 p.m. until dawn.Strong acoustic signals reflected from the dense Chaoborus population overshadowed the fish signals on the echograms, so it is difficult to determine fish distribution at that time.At dawn (3:30 a.m.) Chaoborus larvae resided lower than fish in the epilimnion.Then it descended to hypolimnetic waters when the light intensity increased.Fish moved deeper in the epilimnion, grouped together in schools or likely migrated toward the littoral zone in fear of predation by piscivorous fish or birds.At 9:30 a.m.Chaoborus larvea remained in the deep hypolimnion and its vertical distribution circled back to resemble the situation at 1:30 p.m.

ALAN effect
Plankton samples analysis Analysis of the zooplankton samples revealed that the majority of Chaoborus individuals were in the IVth larval stage of development during our study in the lake with larval density reaching 703.6 ± 87.2 ind.× m −2 and 28.1 ± 3.5 ind.× m −3 (mean ± 1SE).Statistical analysis of zooplankton densities showed that the total abundance of Chaboborus larvae did not differ between the light conditions (Table 1), while it varied significantly between depths (Table 1; Table 1 in the Supplementary Material).The interactions between the two factors were significant (Table 1), indicating that light conditions affected Chaoborus abundance differently at different depths.During the day, Chaoborus density was significantly higher in the deep, dark and anoxic layers of the lake (15-20 and 20-25 m) compared to the upper layers (0-5, 5-10 and 10-15 m) (Fig. 3; Table 2 in the Supplementary Material).At night, before exposure to ALAN, we observed an inverse distribution of Chaoborus in the lake compared to the daytime period.Chaoborus abundance was significantly higher in upper, warm and oxygenated layers (0-5, 5-10 m) than in lower lake layers (10-15, 15-20 and 20-25 m) (Fig. 3; Table 2 and 3 in the Supplementary Material).During ALAN exposure Chaoborus abundance was the highest in the middle water layers (5-10 and 10-15 m), slightly lower in the surface layer (0-5 m), and lowest in the deepest layers (15-20, 20-25 m) (Fig. 3; Table 2 in the Supplementary Material).Chaoborus abundance during ALAN exposure was significantly higher in the upper layers (0-5, 5-10, 10-15 m) and lower in the deepest layers (15-20, 20-25 m) compared to daytime (Fig. 3; Table 3 in the Supplementary Material), while it was significantly lower in the 0-5 m surface layer and much higher in the 10-15 m middle layer compared to nighttime (Fig. 3; Table 3 in the Supplementary Material).After exposure to ALAN, the highest abundance of Chaoborus was found in the 5-10 m layer, slightly lower but      significantly different in the 0-5 and 10-15 m layers, while Chaoborus was virtually absent in the deepest layers (15-20, 20-25 m) devoid of oxygen (Fig. 3; Table 2 in the Supplementary Material).However, we did not observe any significant differences in Chaoborus abundance for any depth between ALAN and darkness 1 h after turning off the light (Fig. 3; Table 3 in the Supplementary Material).
ALAN did not seem to affect the vertical distribution of pelagic crustaceans-potential Chaoborus prey (Fig. 1 in the Supplementary Material).

Sonar recordings analysis
The sonar recordings made it possible to track the distribution of Chaoborus larvae in more detail.The sonar graph indicates that at midnight most larvae were in the warm, oxygenated epilimnion between 0 and 6 m, with few below (Fig. 2).Minutes after the light was switched on, we could observe below the lamp fast descending of the larvae along with an increasing number of fish appearing above them (Fig. 2).Ten minutes after the light phase began, Chaoborus larvae could hardly be detected on the echograms between 0 and 5 m, with some of them observed between 5 and 9 m and most of them between 9 and 13 m in the hypoxic zone.Few individuals  www.nature.com/scientificreports/were visible below 13 m in the anoxic zone at that time.Over time of illumination the density of fish increased and they began to form shoals in subsurface water.The cross sectional echogram taken by the cruising boat 2 m away from the lamp during the ALAN phase revealed a relatively narrow range of spatial effect of the applied light on the Chaoborus distribution.The artificial light affected distribution of Chaoborus larvae within 12 m radius from the lamp approximately (Fig. 5).With the light off, the schools of fish dispersed almost immediately and their density decreased below the lamp as they lost the attractant point.At the same time, Chaoborus gradually began to rise to the surface (Fig. 2).The upward movement of Chaoborus after the light was turned off seemed slower than the downward one after the light was switched on (Fig. 2).

C. flavicans larvae tolerance to anoxic conditions
Survival analysis revealed that survivorship of C. flavicans larvae in the anoxic conditions was lower compared to normoxic ones (Fig. 6; Fig. 2 in the Supplementary Material).Only 4% of all tested larvae stayed alive under 72 h of anoxic conditions compared to 92% in the normoxic ones at the temperature of 9 °C (Fig. 6).The best-fit logistic regression model for the data on mortality of Chaoborus larvae (M) as a function of time in anoxia was empirically estimated as M = 0.98375/(1 + 40.86260 × exp(− 0.09741 × T)), where M is the mortality rate and T is the time in hours in anoxia.According to the logistic regression model, the LT50 of Chaoborus larvae in anoxia (indicating the time of anoxia that causes 50% mortality in experimental animals) was 38 h 25 min.
A few hours before physiological death the larvae used to appear at the water surface in the experimental chambers (Fig. 6).Survival analysis indicated that the prevalence of Chaoborus larvae on the water surface over time differed statistically between normoxic and anoxic conditions (Fig. 6; Fig. 3 in the Supplementary Material).The hazard ratio expressing effect size was significantly higher under anoxia than normoxia assumed as the  www.nature.com/scientificreports/reference (Fig. 6; Fig. 3 in the Supplementary Material).Half of the tested population appeared at the water surface in experimental chambers after 22 h and 9 min of anoxic conditions.The best-fit logistic regression model for the data on their surface occurrence (S) as a function of time in anoxia was empirically estimated as S = 1.04029/(1 + 25.12443 × exp(− 0.14216 × T)) where S is the surface prevalence rate and T is the time in anoxia in hours.

Discussion
Our study evidenced a strong effect of artificial light on the distribution of fish and Chaoborus in the middle of a lake at night.Within seconds of turning on the light, young fish began to appear in the vicinity of the lamp, and their density increased rapidly (Fig. 2).Within minutes, the incoming fish began to school, as they typically do during daylight hours (Fig. 1), probably as a defense mechanism against piscivorous fish that may have also been attracted by the artificial light 37 .A number of fish dispersed into the deeper and darker epilimnetic waters (Figs. 2, 5), where they probably continued to feed on zooplankton.Unfortunately, the determination of fish identity, density and their foraging pressure was not within the scope of our study.Our results are consistent with previously published findings on the effects of ALAN on fish behavior in pelagic waters 36,37 .
A novel finding of our study is the strong effect of ALAN on the distribution of Chaoborus.When exposed to artificial light, unlike fish, Chaoborus larvae rapidly descended from the resource-rich epilimnion to the cooler, hypoxic metalimnion and remained there until the light was turned off (Figs. 2, 3), probably as a defense against the increased risk of fish predation.Downwards movements of Chaoborus larvae were considerably faster than upward movements after the light was turned off (Fig. 2), which could be interpreted as a cautious response to anticipated surface risk.Chaoborus larvae descended a few meters into poorly lit, hypoxic metalimnetic waters presumably in response to the risk of fish predation increased by ALAN.Interestingly, only a small fraction of larvae were observed below 13 m (below the oxycline but still in the metalimnion) under ALAN conditions (Figs. 2, 3), while most of them were found much deeper during the daytime hours (Figs. 1, 3).Why did most of the Chaoborus not migrate deeper into the safer hypolimnetic water under artificial light, where fish rarely venture?Could they feel safe in metalimnetic waters due to low light intensity under ALAN conditions or low oxygen levels which should reduce fish predation pressure?It seems unlikely.First the larvae under ALAN conditions resided in metalimnetic water at two orders of magnitude higher light intensity (1.4 × 10 −2 ± 0.8 × 10 −2 μmol × m −2 × s −1 at mean population depth) compared to their daytime residence depth in anoxic hypolimnetic refugia (1.1 × 10 −4 ± 0.8 × 10 −4 μmol × m −2 × s −1 at mean population depth).In addition, Chaoborus experienced/'tolerated' two orders of magnitude greater variability in light conditions under artificial light at night than during daylight hours.Finally, fish are able to temporarily dive into oxygen-deficient waters in complete darkness in search of food 47,48 .Therefore, Chaoborus should not have felt safe in metalimnion under ALAN conditions.What could prevent Chaoborus from hiding in the deeper and safer strata under ALAN conditions?Risky residence in metalimnetic waters is unlikely to be driven by the need to reduce the energetic costs associated with long-range vertical movement, as these costs appear to be marginal due to the adoption of a hydrostatic mechanism of vertical migration 49 .These might include vertical gradients in temperature, food concentration or oxygen level that are negatively correlated with depth.The slower metabolic rate caused by prolonged residence in deeper, colder waters might not allow Chaoborus to complete its life cycle in a given season, under ALAN conditions.The vertical distribution of food could also influence larvae to restrict their descent into the deeper layers of a lake.Chaoborus larvae are known to forage near the water surface during dark nights 50,51 .However, further studies are needed to verify the significance of this hypothesis.
The vertical distribution of food could also force larvae to limit their descent into the deep layers of a lake.Chaoborus larvae are known to forage near the water surface at night 49 in complete darkness 51 .Staying deeper in the water column during the night triggered by artificial illumination may have had an impact on its foraging success not because of lower capture efficiency in the dark, but because of the fewer prey it encountered in deeper waters (Fig. 1 in the Supplementary Material).The avoidance of anoxic layers at night seems, however, to be more important than other reasons for the metalimnetic residence of Chaoborus under ALAN conditions.Some existing data showed that Chaoborus larvae can tolerate anoxic conditions for hours 26,30 rather than days or weeks suggested by others 27,28 .The cyclic nocturnal migration of the closely related C. crystallinus and C. punctipennis from deep anoxic to normoxic surface waters has been suggested to be driven by the need to inactivate toxic residual metabolites accumulated during daily anoxic metabolism and to "repay the oxygen debt" 26,30 .In order to test if similar constraints may limit the range of vertical migration of C. flavicans in our study, we conducted a laboratory assay that revealed limited tolerance of experimental larvae to anoxia.The data show that, the majority of the most hypoxia-tolerant stage IVth larvae did not survive over 38 h of anoxic conditions (Fig. 6).These temporal limits may be even shorter if we consider the much earlier appearance of the majority of experimental larvae at the water surface in (short) experimental vessels (after 22 h of anoxia), which could cause their ecological mortality (consumption by fish) under ALAN conditions in the field.All this implies that active C. flavicans larvae, like C. cristalinus, must visit oxygenated waters each night to 'pay' the oxygen debt.While the temperature and food gradients are likely to be important, the vertical gradient of predation intensity and oxygen distribution appear to be more critical than others.The gradient of risk of death due to predation and anoxia have an immediate, strong, and direct effect on the fitness of Chaoborus explaining its metalimnetic residence under ALAN conditions.
Unlike Chaoborus, no similar or opposite changes in the vertical distribution of planktonic crustaceans were observed during the analysis of the plankton samples (Fig. 1 in the Supplementary Material).These results are rather surprising, as 7  www.nature.com/scientificreports/crustaceans were either absent or undetectably small in our study due to the low resolution of the plankton sampling technique used.Due to the relatively low height of the lamp used, resulting in a low angle of incidence and high reflection from the water surface, the spatial effect of the ALAN on Chaoborus larvae distribution was not extensive in our tests.The light only affected Chaoborus distribution within a 15 m radius of the lamp used (Fig. 5).It is likely that a more powerful lamp, mounted higher above the water level and shining for a longer period, could have a more widespread and profound effect than observed in our study.It is likely that constant exposure to high levels of illumination could intensify fish predation pressure and exterminate Chaoborus from light-polluted sites, which in turn could alter the composition of the local community of its prey.
For the above reasons we recommend caution when using ALAN in open water to reduce the anthropogenic impact on aquatic communities.Low power lamps, light range limiters or safe light colors can be considered as countermeasures.The use of red light, which is barely visible to most aquatic organisms 52,53 but visible to humans, may be an option.

Study site
The field study was carried out, at different periods of the day, in the deepest basin (31 m) of lake Roś (Great Masurian Lakes, Poland, Europe; 53.67°N, 21.92°E; Fig. 4 in the Supplementary Material) during summer stratification.Roś is a lowland, postglacial, eutrophic (with 1-2 m transparency of water in summer), dimictic, flow-through lake with an average depth of 8.1 m and an area of 189 ha with a 25% littoral covered mainly reeds.The pelagic zone of this lake at night in summer was occupied by planktivorous fry of smelt (Osmerus eperlanus; L., 1758), perch (Perca fluviatilis; L., 1758) and roach (Rutilus rutilus; L., 1758) in the mid-eighties of the twentieth century 54 when the lake was still mesotrophic.Recent published data on pelagic fish composition are not available, but our observations indicate that small perch and bleak (Alburnus alburnus; L., 1758) have recently been the most abundant fish in the open water at night (unpublished data).

Measurements of environmental conditions
Temperature and oxygen concentrations at various depths (Fig. 2) were measured during the tests using a submersible optical dissolved oxygen probe (YSI ProODO).Light intensity at different depths marked on the graphs was measured under the lamp during the experiment at night and day using a portable light meter (LiCor LI-250A) with a spherical underwater PAR quantum sensor (LiCor LI-193R) and on a few occasions for comparison using a commercial portable non-submersible Luxometer (Standard Instruments ST-1308 Light Meter; more details at thecalibrationcentre.co.uk) at the water surface.

Assessment of diurnal natural distribution of fish and Chaoborus
The swim bladders of planktivorous fish and the gas sacs of the phantom midge strongly reflect sound waves in the water, which makes them detectable by sonars 55 .In the present study, a commercial fishing sonar (Lowrance LMS-337c) with a 200 kHz transducer mounted on a cruising boat was used to monitor the spatial distribution of fish and Chaoborus larvae over the course of a day in the main basin of the lake along a single transect extending from the littoral zone to the deepest spot and back on 20-22 June 2015 (Fig. 1).

ALAN effect on vertical distribution of aquatic animals in the pelagic zone
A street lamp with High Pressure Sodium (HPS) bulb of medium power (70 W) (luminaire: FORT MTH-413/70W-B, Kanlux; OSRAM) was used to assess effect of artificial light at night on vertical distribution of pelagic animals.HPS lamps are slowly replacing by LEDs, however in Poland HPS are still widely used.The light spectrum of the lamp is shown on the graph (Fig. 5 in the Supplementary Material).The lamp was mounted on a rail on the side of a boat, relatively low-1.5 m above the water, which "compensated" for its lower power compared to the 250-400 W street lamps more commonly used and mounted at a higher level.The boat with the lamp was double anchored near the deepest spot of the lake far from illuminated settlements (Fig. 4 in the Supplementary Material).The maximum ALAN intensity during the experiments reached 250-450 lx under the lamp at the water surface when measured with the non-submersible luxometer, while 3.3-5.9µmol × m −2 × s −1 when measured approximately 3 cm below the water surface with the PAR underwater quantum sensor.While these light levels were higher than that documented along the seacoast by 56 , it can be experienced near brightly lit marinas or floating restaurants (unpublished data).The vertical distribution of phantom midge below the lamp was determined upon direct zooplankton catches and, in addition, using sonar (Lowrance ELITE-Ti with 200 kHz transducer) on five summer nights between 4 and 25 July 2019.At that time, the sun set at 8.30 p.m. and rose at 4.30 a.m.Moonlight periodicity was much more variable in that period.Max moon light intensity (0.3 lx) 57,58 is 3-4 orders of magnitude lower than the artificial light we used in our tests, so it should have a rather marginal effect on the results of our study (additional information in the Table 4 in the Supplementary Material).Night work was carried out using dim red headlamps, barely visible by aquatic animals 52,53 .At midnight, before switching on the light, zooplankton samples were collected at five subsequent water layers (one sample at each layer) in descending order (5-0 m, 10-5 m, 15-10 m, 20-15 m, 25-20 m) using a quantitative closing plankton net with an opening diameter of 28 cm and a mesh size of 150 µm.Shortly afterwards, an echo sounder was switched on to provide a continuous record of sound reflecting aquatic animals (Chaoborus larvae and fish).After 10 min of echo sounding in natural darkness, the lamp was turned on for an hour and echo sounding continued.At the end of the light period, depth-stratified zooplankton samples were collected in a similar way as before.In three of five experimental nights echo sounding was continued for 1 hour after the light was switched off and stratified zooplankton samples were collected again in the dark.Moreover, at five www.nature.com/scientificreports/occasions midday depth-stratified zooplankton samples as well as light intensity were collected in a similar way to determine daytime Chaoborus larvae vertical distribution in the lake upon direct catches during the tests.Zooplankton samples were analyzed under stereomicroscope and planktonic crustaceans were assigned to the lowest possible taxonomic level.The insect larvae (IVth instar) were classified into the species C. flavicans based on a morphological analysis of mandibular teeth and fan bristles, labial blades and anal fan rays [59][60][61] .The density distribution of Chaoborus larvae at different depths, estimated from plankton samples, was then compared with the sonar data and light intensity.The mean depth of the Chaoborus population was calculated on the basis of zooplankton samples according to the following formula (n 1−5 × d 1−5 )/ n 1−5 , where n is the number of Chaoborus larvae collected in each of five (1-5) subsequent vertical strata 5 m long each one, while d is the mean depth of each of five (1-5) vertical strata.Light intensity at the mean depth of the population was then compared between the day and ALAN treatments.Due to the limited sensitivity (0.01 μmol × m −2 × s −1 ) and length (10 m) of the sensor cable of the submersible light meter, light intensity at the mean depth of the population was estimated (in most cases extrapolated) using the light-depth regression from the collected data from the surface layers, which were evaluated individually.

Laboratory test on temporal survival of anoxic conditions by Chaoborus flavicans
The experimental animals were collected by towing a plankton net vertically between the depths of 25 and 0 m in the afternoon of July 2022 in Lake Roś.Prior to use in the experiment, the animals were kept in air saturated lake water for about 3 h.150 C. flavicans larvae of similar size in the IVth stage of development were pre-selected from live plankton samples.The experimental setup (Fig. 7) consisted of 12 polyethylene terephthalate (PET) bottles (of 475 mL volume) filled with 450 mL of the lake water without sediment, submerged in the common water bath of controlled temperature, simulating the daytime thermal conditions faced by Chaoborus larvae in the hypolimnion of Lake Roś at that time (9 ± 0.5 °C).Each experimental bottle was closed with a tight lid supplied with a gas inlet and outlet.The gas inlet was supplied with a gas diffuser immersed 3 cm below the water surface of the bottle.The gas outlet was located above the water level of the bottle and was equipped with a check valve.Ten randomly selected Chaoborus larvae were transferred to each of 10 PET bottles.Two additional bottles without animals were used to periodically measure the oxygen content in the experimental bottles.At the beginning of the experiment, water in six bottles was aerated with an air pump, while in the other six it was vented with nitrogen gas for 3 h.Two hours of water venting with nitrogen proved sufficient to reduce oxygen levels to near-anoxic conditions of about 0.03 mg × L −1 .Both air and nitrogen ventilation of the water were repeated cyclically for 1 h in the morning and evening of the experimental days.The viability of the experimental

Statistical analysis
Statistical analysis was performed in R v. 4.2.3 62 .For all statistical tests, the significance level was set at α = 0.05.
To investigate the effects of treatment and depth and their interaction on the abundance of Chaoborus larvae in the lake, we used two-factor generalized linear mixed models (GLMMs 63 ) with the template model builder (the glmmTMB package v.1.1.3 64).The number of Chaoborus larvae in the plankton samples was set as the response variable, treatment (daytime, nighttime before ALAN, ALAN, nighttime after ALAN) as the first fixed effect, and depth range (0-5, 5-10, 10-15, 15-20, 20-25 m) as the second fixed effect and random intercept.To better fit the data into the model, a quadratic transformation of the response variable was performed.The count data, after the above transformation, were modeled with a Gaussian regression with a zero-inflated parameter.The significance of the interaction between factors was assessed using the analysis of deviance and the Wald type II Chi-squared difference test (χ 2 ) (car package v.3.0-12 65).The model fit to the data was optimized using the stats:anova() function and visual inspection of the DHARMa scaled residual plots (the DHARMA package v.0.4.5 66 ).Post-hoc tests (for both factors and interactions) were performed using planned contrast for estimated marginal means (EMMs; the emmeans package (v.1.7.2 67 ).The 'holm' p value adjustment was applied to control for type I error inflation due to multiple testing.Light intensity at mean depth of the Chaoborus population was compared between the daytime and ALAN conditions using the nonparametric Wilcoxon rank sum test due to unequal variances in the compared data sets.
The effect of time in anoxia on mortality and surface prevalence of laboratory animals was investigated using non-linear least squares (NLS) regression and automatically determined model parameters by using self-starting function (the NLS logistic function in R).The model parameters allowed to estimate LT50-time causing 50% mortality within tested individuals as well as time of appearance of half of the population at the water surface in anoxic conditions.Survival analysis (package "survival" v.3.5-3 68,69) and the nonparametric Kaplan-Maier method for censored data 70 with the log-rank (chi square) test were used to compare survivorship of Chaoborus larvae between oxygen treatments.The effect size was evaluated using the hazard ratio method and the Cox proportional hazards model 71 .

Figure 1 .
Figure 1.The natural diurnal pattern of spatial distribution of C. flavicans larvae and fish in the main basin of Lake Roś in July 2015.White color indicate water, while warm colors indicate sound reflecting objects in the water.Black arrows indicate the presence of fish, "Ch" indicates the presence of Chaoborus larvae.A single transect extending from the littoral zone to the deepest point and back.Vertical gradients of temperature (measured in °C) and oxygen (measured in mg × L −1 ) along the water depth in the lake are shown in the lower left corner.

Figure 2 .
Figure 2. Example of temporal dynamics of vertical distribution of Chaoborus flavicans and fish under the lamp before, during and after artificial illumination of the pelagic zone in Lake Roś (on the right-"Ch"-presence of Chaoborus; white arrows point to fish).The grey dashed line represents the beginning of the anoxic layer.Left panel shows the vertical distribution of artificial light and physicochemical parameters during the ALAN phase in Lake Roś (O 2 : oxygen concentration (mg × L −1 ); T: temperature (°C); Light: photon flux (μmol × m −2 × s −1 ).Photon flux corresponds to ALAN treatment only.

Figure 3 .
Figure 3. Vertical distribution of Chaoborus larvae (mean proportion ± 1SE) during the day, at night before ALAN, at night during ALAN and at night after ALAN.

Figure 4 .
Figure 4. Mean light intensity (± 1SE) at mean (± 1SE) depth of the Chaoborus population during daytime compared to ALAN conditions in Lake Roś.

Figure 5 .
Figure 5. Cross-sectional echogram recorded 30 min after the light was turned on.Light bulb indicates the location of an artificial light source.White arrows indicate the presence of fish, "Ch" indicates the presence of Chaoborus larvae.

Figure 6 .
Figure 6.Effect of time on mortality of Chaoborus larvae in anoxic (open red circles) and normoxic (open blue circles) conditions with logistic regressions (solid lines in corresponding color) fitted to the experimental data.Prevalence of experimental animals at the water surface over time is shown with plus marks in anoxic (red) and normoxic (blue) conditions.The dashed line shows the logistic regression fitted to the data on surface appearance in anoxic conditions.No logistic regression could be fitted to the surface appearance data in the normoxic conditions.

Figure 7 .
Figure 7. Scheme of the experimental setup on C. flavicans larvae tolerance to anoxic conditions.

Table
Table of the deviance analysis of the generalized linear mixed effects model to test the effect of depth and treatments and their interaction on the abundance of Chaoborus larvae in the lake.Statistically significant differences (α = 0.05) are shown in bold.χ 2 , Chi-squared test; df, Degrees of freedom, p, p value.
animals was assessed every 6 h in each bottle.The criterion for their physiological death was lack of locomotor activity which was typically preceded with surface appearance of barely mobile individuals.The experiment was continued for 72 h, when most of the animals in the anoxic treatment appeared immobile.Experiment complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.Due to the lack of clear sex-dependent morphological characteristics, the sex of Chaoborus larvae was not determined in either the field study or the laboratory experiment.